Age-Dependent Expression of Humanin in the Bull (Bos Taurus) Testis and Its Potential Role in Regulating Hormonal Status, Oxidative Stress and MicroRNA Expression
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Abstract
Humanin (HN), a mitochondrial-derived peptide with cytoprotective functions, has not been thoroughly investigated in the bull gonad. This study characterized the developmental localization of HN in the bull testis and its relationship with hormonal status, oxidative stress, and key miRNAs. An age dependent HN expression pattern was seen by immunofluorescence examinations of testicular tissues from pre-pubertal, puberty, mature, and aged bulls. So that, pre-pubertal and aged groups showed faint HN immunoreactivity, pubertal animals showed moderate immunoreactivity, and mature adults showed the most intense immune response (P ≤ 0.0001). In contrast to this, miR-202-5p and miR-21 had their highest expression levels during pre-pubertal, pubertal, and mature testes and a significant decrease in aged bulls (P ≤ 0.0001). Despite the contrasting developmental trends observed at the group level, a correlation analysis involving all individual animals (n = 40) demonstrated a robust positive association between HN and miR-202-5p levels (r = 0.8196, P < 0.001). This finding implies a close regulatory connection between these molecules that functions throughout various developmental stages. Quantitatively, HN levels was correlated positively with serum testosterone (r = 0.5852, P < 0.01) and the antioxidant enzyme SOD (r = 0.8208, P < 0.001), and negatively with the oxidative stress marker MDA (r = - 0.7140, P < 0.001). The current findings show the first developmentally-specific HN expression in the bull testis, which is linked to peak reproductive maturity, hormonal balance, and redox homeostasis. The correlation with miR-202-5p suggests that there is a potential new regulatory network, and that HN is a crucial peptide for testicular function and aging. The results of this study could facilitate additional investigations into the distribution of HN throughout gonadal development, ultimately enhancing the reproductive efficiency of livestock.
Received: 27 September 2025
Revised: 18 October 2025
Accepted: 30 November 2025
Published : 28 Desember 2025
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1. Acevedo-Rodriguez A, Kauffman AS, Cherrington BD, Borges CS, Roepke TA, Laconi M. Emerging insights into hypothalamic-pituitary-gonadal axis regulation and interaction with stress signaling. J Neuroendocrinol. 2018;30(10):e12590. https://doi.org/10.1111/jne.12590 DOI: https://doi.org/10.1111/jne.12590
2. Mohammed BT, Donadeu FX. Localization and in silico-based functional analysis of miR-202 in bull testis. Reprod Domest Anim. 2022;57(9):1082-1087. https://doi.org/10.1111/rda.14159 DOI: https://doi.org/10.1111/rda.14159
3. Dong S, Chen C, Zhang J, Gao Y, Zeng X, Zhang X. Testicular aging, male fertility and beyond. Front Endocrinol. 2022;13:1012119.
https://doi.org/10.3389/fendo.2022.1012119 DOI: https://doi.org/10.3389/fendo.2022.1012119
4. Matzkin ME, Calandra RS, Rossi SP, Bartke A, Frungieri MB. Hallmarks of Testicular Aging: The challenge of anti-inflammatory and antioxidant therapies using natural and/or pharmacological compounds to improve the physiopathological status of the aged male gonad. Cells. 2021;10(11):3114. https://doi.org/10.3390/cells10113114 DOI: https://doi.org/10.3390/cells10113114
5. Kandil NT, Sabry DA, Ashry NI, El-Sayyad HI. Studies on the anti-aging properties of bovine whey supplementation on reproductive hormonal profiles and sperm structure and function of adult and senile rats. Food Biosci. 2022;45:101369.
https://doi.org/10.1016/j.fbio.2021.101369 DOI: https://doi.org/10.1016/j.fbio.2021.101369
6. Matsuoka M, Hashimoto Y, Aiso S, Nishimoto I. Humanin and colivelin: neuronal-death-suppressing peptides for Alzheimer's disease and amyotrophic lateral sclerosis. CNS Drug Rev. 2006;12(2):113-122. https://doi.org/10.1111/j.1527-3458.2006.00113.x DOI: https://doi.org/10.1111/j.1527-3458.2006.00113.x
7. Lue Y, Swerdloff R, Liu Q, Mehta H, Hikim AS, Lee KW, et al. Opposing roles of insulin-like growth factor binding protein 3 and humanin in the regulation of testicular germ cell apoptosis. Endocrinol. 2010;151(1):350-357. https://doi.org/10.1210/en.2009-0577 DOI: https://doi.org/10.1210/en.2009-0577
8. Rao M, Wu Z, Wen Y, Wang R, Zhao S, Tang L. Humanin levels in human seminal plasma and spermatozoa are related to sperm quality. Androl. 2019;7(6):859-866. https://doi.org/10.1111/andr.12614 DOI: https://doi.org/10.1111/andr.12614
9. Zhu S, Hu X, Bennett S, Xu, Mai Y. The molecular structure and role of humanin in neural and skeletal diseases, and in tissue regeneration. Front. Cell Dev. Biol. 2022;10:823354. https://doi.org/10.3389/fcell.2022.823354 DOI: https://doi.org/10.3389/fcell.2022.823354
10. Xia Y, Zhang HY, Ma S, Zhou F. Age-related changes in humanin expression in the ovarian tissue of rat. Curr Med Sci. 2023;43(3):579-584. https://doi.org/10.1007/s11596-023-2732-7 DOI: https://doi.org/10.1007/s11596-023-2732-7
11. Katiyar R, Ghosh SK, Kumar A, Pande M, Gemeda AE, Rautela R, et al. Cryoprotectant with a mitochondrial derived peptide, humanin, improves post-thaw quality of buffalo spermatozoa. Cryo Letters. 2022;43(1):32-41. https://doi.org/10.54680/fr22110110212 DOI: https://doi.org/10.54680/fr22110110212
12. Katiyar R, Ghosh SK, Karikalan M, Kumar A, Pande M, Gemeda AI, et al. An evidence of humanin-like peptide and humanin mediated cryosurvival of spermatozoa in buffalo bulls. Theriogenology. 2022;194:13-26. https://doi.org/10.1016/j.theriogenology.2022.09.013 DOI: https://doi.org/10.1016/j.theriogenology.2022.09.013
13. Chen J, Gao C, Lin X, Ning Y, He W, Zheng C, et al. The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis. Development. 2021;148(24):dev199799. https://doi.org/10.1242/dev.199799 DOI: https://doi.org/10.1242/dev.199799
14. Salilew-Wondim D, Gebremedhn S, Hoelker M, Tholen E, Hailay T, Tesfaye D. The role of microRNAs in mammalian fertility: from gametogenesis to embryo implantation. Int J Mol Sci. 2020;21(2):585. https://doi.org/10.3390/ijms21020585 DOI: https://doi.org/10.3390/ijms21020585
15. Zhang J, Liu W, Jin Y, Jia P, Jia K, Yi M. MiR-202-5p is a novel germ plasm-specific microRNA in zebrafish. Sci Rep. 2017;7(1):7055.
https://doi.org/10.1038/s41598-017-07675-x DOI: https://doi.org/10.1038/s41598-017-07675-x
16. Ding Q, Jin M, Wang Y, Liu J, Kalds P, Wang Y, et al. Transactivation of miR-202-5p by steroidogenic factor 1 (SF1) induces apoptosis in goat granulosa cells by targeting TGFβR2. Cells. 2020;9(2):445. https://doi.org/10.3390/cells9020445 DOI: https://doi.org/10.3390/cells9020445
17. Koenig EM, Fisher C, Bernard H. The beagle dog MicroRNA tissue atlas: identifying translatable biomarkers of organ toxicity. BMC Genomics. 2016;17(649). https://doi.org/10.1186/s12864-016-2958-x DOI: https://doi.org/10.1186/s12864-016-2958-x
18. Jia KT, Zhang J, Jia P, Zeng L, Jin Y, Yuan Y, et al. Identification of microRNAs in zebrafish spermatozoa. Zebrafish. 2015;12(6):387-397.
https://doi.org/10.1089/zeb.2015.1115 DOI: https://doi.org/10.1089/zeb.2015.1115
19. Hu R, Xu Y, Han B, Chen Y, Li W, Guan G, et al. MiR-202-3p determines embryo viability during mid-blastula transition. Front Cell Dev Biol. 2022;10:897826. https://doi.org/10.3389/fcell.2022.897826 DOI: https://doi.org/10.3389/fcell.2022.897826
20. Dabaja AA, Mielnik A, Robinson BD, Wosnitzer MS, Schlegel PN, Paduch DA. Possible germ cell-Sertoli cell interactions are critical for establishing appropriate expression levels for the Sertoli cell-specific MicroRNA, miR-202-5p, in human testis. Basic Clin Androl. 2015;25:2.
https://doi.org/10.1186/s12610-015-0018-z DOI: https://doi.org/10.1186/s12610-015-0018-z
21. Yuan M, Yang X, Duscher D, Xiong H, Ren S, Xu X, et al. Overexpression of microRNA-21-5p prevents the oxidative stress-induced apoptosis of RSC96 cells by suppressing autophagy. Life Sci. 2020;256:118022. https://doi.org/10.1016/j.lfs.2020.118022 DOI: https://doi.org/10.1016/j.lfs.2020.118022
22. Yu P, Zhao X, Zhou D, Wang S, Hu Z, Lian K, et al. The microRNA-mediated apoptotic signaling axis in male reproduction: a possible and targetable culprit in male infertility. Cell Biol Toxicol. 2025;41(1):54. https://doi.org/10.1007/s10565-025-10006-w DOI: https://doi.org/10.1007/s10565-025-10006-w
23. Tang Q, Zhang Y, Yue L, Ren H, Pan C. Ssc-MiR-21-5p and Ssc-MiR-615 regulates the proliferation and apoptosis of Leydig cells by targeting SOX5. Cells. 2022;11(14):2253. https://doi.org/10.3390/cells11142253 DOI: https://doi.org/10.3390/cells11142253
24. Rajak SK, Kumaresan A, Gaurav MK, Layek SS, Mohanty TK, Muhammad Aslam MK, et al. Testicular cell indices and peripheral blood testosterone concentrations in relation to age and semen quality in crossbred (holstein Friesian × tharparkar) bulls. Asian-Australas J Anim Sci. 2014;27(11):1554-1561. https://doi.org/10.5713/ajas.2014.14139 DOI: https://doi.org/10.5713/ajas.2014.14139
25. Li EZ, Li DX, Zhang SQ, Wang CY, Zhang XM, Lu JY, et al. 17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice. Asian J Androl. 2007;9(5):659-667. https://doi.org/10.1111/j.1745-7262.2007.00288.x DOI: https://doi.org/10.1111/j.1745-7262.2007.00288.x
26. Mercati F, Guelfi G, Martì MJI, Dall'Aglio C, Calleja L, Caivano D, et al. Seasonal variation of NGF in seminal plasma and expression of NGF and its cognate receptors NTRK1 and p75NTR in the sex organs of rams. Domest Anim Endocrinol. 2024;89:106877.
https://doi.org/10.1016/j.domaniend.2024.106877 DOI: https://doi.org/10.1016/j.domaniend.2024.106877
27. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem.1979;95:351-358.
https://doi.org/10.1016/0003-2697(79)90738-3 DOI: https://doi.org/10.1016/0003-2697(79)90738-3
28. Chin YP, Keni J, Wan J, Mehta H, Anene F, Jia Y, et al. Pharmacokinetics and tissue distribution of humanin and its analogues in male rodents. Endocrinol. 2013;154(10):3739-3744. https://doi.org/10.1210/en.2012-2004 DOI: https://doi.org/10.1210/en.2012-2004
29. Lei H, Rao M. The role of humanin in the regulation of reproduction. Biochim Biophys Acta Gen Subj. 2022;1866(1):130023.
https://doi.org/10.1016/j.bbagen.2021.130023 DOI: https://doi.org/10.1016/j.bbagen.2021.130023
30. Jia Y, Lue YH, Swerdloff R, Lee KW, Cobb LJ, Cohen P, et al. The cytoprotective peptide humanin is induced and neutralizes Bax after pro-apoptotic stress in the rat testis. Andrology. 2013;1(4):651-659. https://doi.org/10.1111/j.2047-2927.2013.00091.x DOI: https://doi.org/10.1111/j.2047-2927.2013.00091.x
31. Yen K, Mehta HH, Kim SJ, Lue Y, Hoang J, Guerrero N, et al. The mitochondrial derived peptide humanin is a regulator of lifespan and healthspan. Aging (Albany NY). 2020;12(12):11185-11199. https://doi.org/10.18632/aging.103534 DOI: https://doi.org/10.18632/aging.103534
32. Karachaliou CE, Livaniou E. Neuroprotective action of humanin and humanin analogues: research findings and perspectives. Biol. (Basel). 2023;12(12):1534. https://doi.org/10.3390/biology12121534 DOI: https://doi.org/10.3390/biology12121534
33. Wang Z, Li D, Zhou G, Xu Z, Wang X, Tan S, et al. Deciphering the role of reactive oxygen species in idiopathic asthenozoospermia. Front Endocrinol. 2025;16:1505213. https://doi.org/10.3389/fendo.2025.1505213 DOI: https://doi.org/10.3389/fendo.2025.1505213
34. Muzumdar RH, Huffman DM, Atzmon G, Buettner C, Cobb LJ, Fishman S, et al. Humanin: a novel central regulator of peripheral insulin action. PLoS One. 2009;4(7):e6334.
https://doi.org/10.1371/journal.pone.0006334 DOI: https://doi.org/10.1371/journal.pone.0006334
35. Coradduzza D, Congiargiu A, Chen Z, Cruciani S, Zinellu A, Carru C, et al M. Humanin and its pathophysiological roles in aging: a systematic review. Biol. (Basel). 2023;12(4):558. https://doi.org/10.3390/biology12040558 DOI: https://doi.org/10.3390/biology12040558
36. Garza S, Sottas C, Gukasyan HJ, Papadopoulos V. In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function. Front Toxicol. 2024;6:1357857. https://doi.org/10.3389/ftox.2024.1357857 DOI: https://doi.org/10.3389/ftox.2024.1357857
37. Lucas-Herald AK, Mitchell RT. Testicular Sertoli cell hormones in differences in sex development. Front Endocrinol. 2022;13:919670.
https://doi.org/10.3389/fendo.2022.919670 DOI: https://doi.org/10.3389/fendo.2022.919670
38. Onat E, Kocaman N, Hancer S. The protective effects of humanin in rats with experimental myocardial infarction: The role of asprosin and spexin. Heliyon. 2023;9(8):e18739. https://doi.org/10.1016/j.heliyon.2023.e18739 DOI: https://doi.org/10.1016/j.heliyon.2023.e18739
39. Niu Z, Goodyear SM, Rao S, Wu X, Tobias JW, Avarbock MR, et al. MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A. 2011;108(31):12740-12745. https://doi.org/10.1073/pnas.1109987108 DOI: https://doi.org/10.1073/pnas.1109987108
40. Chen J, Cai T, Zheng C, Lin X, Wang G, Liao S, et al. MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins. Nucleic Acids Res. 2017;45(7):4142-4157. https://doi.org/10.1093/nar/gkw1287 DOI: https://doi.org/10.1093/nar/gkw1287
41. Werry N, Russell SJ, Gillis DJ, Miller S, Hickey K, Larmer S, et al.Characteristics of miRNAs present in bovine sperm and associations with differences in fertility. Front Endocrinol (Lausanne). 2022;13:874371. https://doi.org/10.3389/fendo.2022.874371 DOI: https://doi.org/10.3389/fendo.2022.874371
42. Sass S, Dietmann S, Burk UC, Brabletz S, Lutter D, Kowarsch A, et al. MicroRNAs coordinately regulate protein complexes. BMC Syst Biol. 2011;5:136. https://doi.org/10.1186/1752-0509-5-136 DOI: https://doi.org/10.1186/1752-0509-5-136